The unexpected demonstration that the gene encoding the Krebs cycle enzyme fumarate hydratase conforms to the classical genetic model of a tumor suppressor, predisposing individuals carrying germline mutations to cancers bearing somatic inactivation of the second allele (Tomlinson et al., 2002
), has raised great interest in defining the associated oncogenic pathway(s).
Structural, biochemical, and biological analyses have established that fumarate, which accumulates in FH-defective cells, binds to PHDs and inhibits their catalytic activity leading to upregulation of HIF transcriptional pathways, as occurs in hypoxia (Hewitson et al., 2007; Koivunen et al., 2007
). Hypoxia and activation of the HIF system are commonly associated with aggressive cancer (Harris, 2002
), but despite intense investigation, cause and effect have remained difficult to distinguish. Since HIF activation is a direct consequence of inactivation of the FH tumor suppressor, irrespective of hypoxia, this link might indicate causality. Indeed, striking activation of HIF was observed in the mouse model described above and in FH-associated human cancer, as well as in tumors linked to inactivation of the succinate dehydrogenase enzyme complex and mutations in genes encoding isocitrate dehydrogenases 1 and 2, which have also been defined as tumor suppressors or oncogenes (Pollard and Ratcliffe, 2009
Our findings clearly demonstrate that despite striking activation of Hif and a number of Hif-target genes in Fh1-deficient cells, neither upregulation of Hif nor inactivation of the Phds is required or responsible for the hyperplastic cystic phenotype observed in the mouse model. Surprisingly we found that, rather than ameliorating cyst development, combined inactivation of Hif-1α (but not Hif-2α) and Fh1, greatly exacerbated cystic hyperplasia. Thus, in this setting, upregulation of Hif-1α appears to exert an antiproliferative effect. While this is apparently at odds with the frequently observed upregulation of HIF-1α in cancer, it is consistent with emerging evidence for differential effects of HIF-1α and HIF-2α in tumor biology. HIF-1α antagonizes MYC function, whereas HIF-2α promotes MYC activity (Gordan et al., 2007
), and overexpression of HIF-1α and HIF-2α have contrasting effects on the growth of experimental tumors from VHL-defective RCC lines (Raval et al., 2005
). Furthermore, mutational analyses reveal a modest but significant prevalence of HIF-1α inactivating mutations in VHL-associated clear cell RCC (Dalgliesh et al., 2010; Morris et al., 2009; Shen et al., 2011
). Nevertheless, the finding that inactivation of Hif-2α had no effect on Fh1-associated cystic disease either alone, or in combination with Hif-1α inactivation, differs from findings reported in a similar mouse model of VHL-associated renal neoplasia. In this latter model, combined inactivation of Arnt, but not Hif-1α, ameliorated Vhl-associated renal cystic disease, implying that Hif-2α might be responsible for the cyst development associated with Vhl loss (Rankin et al., 2006
). Hence, we conclude that despite the common activation of HIF pathways in VHL- and FH-associated neoplasia, the oncogenic mechanisms are likely to be different.
Combined inactivation of Fh1 and Hif-1α in our mouse model enabled Fh1 dependent changes in transcript profiles to be interrogated without confounding influences from activation of extensive HIF-dependent transcriptional cascades and revealed striking activation of the Nrf2-mediated antioxidant signaling pathway. Further analyses in cell lines derived from Fh1−/−
MEFs, mouse cystic tissues and FH-associated human cancer demonstrated that activation of the canonical NRF2 antioxidant pathway arose as a direct consequence of FH inactivation. Though we cannot exclude other influences on Nrf2 dysregulation, the demonstration of high levels of succination on critical cysteine residues in KEAP1, the abnormal activity of transfected KEAP1 in Fh1−/−
cells and the maintenance of GSH levels in Fh1−/−
cells all argue that Nrf2 activation results from succination of KEAP1 rather than general oxidant stress, at least under the conditions of these experiments (). NRF2 acts as a master regulator controlling the ability of mammalian cells to adapt rapidly to stress caused by oxidants and electrophiles, through the induction of ARE containing genes (Nguyen et al., 2003
). KEAP1 complexes with Cullin 3 (CUL3) forming an ubiquitin E3 ligase that degrades NRF2. Although not fully understood, the interactions by which KEAP1 controls the levels of NRF2, its cellular localization and transcriptional activity are complex. However, all current models propose that cysteine residues of KEAP1 are modified in response to oxidative stress, resulting in compromised function of the ubiquitin E3 ligase complex that effects proteasomal degradation of NRF2 and enhanced NRF2 stability (Nguyen et al., 2009
Given that succination of multiple proteins occurs in FH-defective cells and tumors (Bardella et al., 2011
), we enquired whether defective KEAP1 function in Fh1 deficient cells might be associated with succination of critical regulatory cysteine residues. MS/MS analysis provided clear evidence of succination on KEAP1 residues Cys151 and Cys288 in association with defective regulation of Nrf2 in Fh1−/−
cells. These two residues are among those cysteines, Cys23, 151, 273, 288, and 613, that are conserved between mouse and human and which have been identified as having functional roles in the activity of Keap1 (Hayes et al., 2010; McMahon et al., 2010; Taguchi et al., 2011
). Transgenic complementation studies have shown that Cys273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo, while Cys151 is important in facilitating Nrf2 activation in studies with MEFs from a Keap1 (C151S) transgenic mouse model (Yamamoto et al., 2008
). Although Cys613 is part of a zinc sensor system (McMahon et al., 2010
) and might be modified by fumarate leading to zinc signaling (Cousins et al., 2006
) our microarray data and pathway analyses do not suggest that this pathway is dysregulated in Fh1-associated cystic disease.
Interestingly, a recent study has described activation of Nrf2 by exogenous fumarate both in vitro and in vivo (Linker et al., 2011
); similar to our findings, they provide direct evidence that KEAP1 is modified at Cys151, though not at Cys288. Whereas this study utilized cell permeable fumaric acid esters (mono- and dimethylfumarate), we have demonstrated that pathophysiological levels of fumarate associated with cancer are sufficient to succinate KEAP1 and activate Nrf2 signaling.
To our knowledge, mutations of KEAP1
, or downstream target genes, which might shed further light on the role of this pathway in FH-associated oncogenesis, have not yet been described in Type 2 pRCC. Given the extensive transcriptional cascade regulated by NRF2, whether and how dysregulation of KEAP1/NRF2 signaling drives oncogenesis requires further investigation. However, both KEAP1
somatic missense mutations have been identified in a variety of tumors. Moreover, functional assays and the clustering of mutations at sites that disrupt KEAP1/NRF2 regulation have suggested that dysfunction of KEAP1 contributes in some way to oncogenesis in these settings (Hayes and McMahon, 2009; Taguchi et al., 2011
). Though KEAP1/NRF2 dysregulation has been considered as an adaptive response that might particularly affect later stages of oncogenesis, recent data in mouse models of pancreatic and lung cancer, where Nrf2 ablation was associated with reduced cellular proliferation, have suggested an early effect (DeNicola et al., 2011
). Our data indicating that Nrf2 dysregulation occurs early in the course of hyperplastic cyst development, as a direct consequence of Fh1 inactivation, are consistent with this possibility.
In summary, our investigations have revealed that despite the striking upregulation of the HIF transcriptional cascade in FH-associated neoplasia, these pathways do not appear to contribute to hyperplastic renal cyst formation, at least in a mouse model that recapitulates many features of the human disease; rather, our findings have raised the possibility of an alternative oncogenic action of fumarate through the activation of antioxidant response pathways by succination of KEAP1, and possibly other proteins with tumor suppressor functions.